The mapping and sequencing of the human genome will probably be one of the most remarkable achievements of the 21st century. This effort, led by the Human Genome Project has created an unprecedented opportunity to characterize and understand the function of the repertoire of newly discovered genes at the individual gene level and the complexities in the interaction of the gene products at the organism level (Austin, C. P, 2004, Annu. Rev. Med. Vol. 55, pp 1-13). Only when this task is truly achieved and understood can the scientific community deliver on the promise of the Human Genome Project for blockbuster drugs and targeted therapy for different ailments afflicting mankind (Austin, C. P, 2004, Annu. Rev. Med. Vol. 55, pp 1-13).
The task of functional characterization of newly discovered genes is initiated by the introduction of plasmids containing a copy of the gene of interest into mammalian cells so that its function can be assessed in an appropriate cellular context such as proliferation, viability, gene expression and differentiation amongst others (Kramer, R. and Cohen D., 2004, Nat. Rev. Drug Discov. Vol. 3, pp 965-972). Alternatively, dominant negative versions of the gene of interest which interfere with the function of the wildtype gene or reagents which down regulate gene expression such as anti-sense oligonucleotides or interfering RNA (RNAi) can also be introduced into mammalian cells and its impact assessed in various cell-based assays described above (Kramer, R. and Cohen D., 2004, Nat. Rev. Drug Discov. Vol. 3, pp 965-972). Regardless of the approach taken the main hurdle in introducing these reagents into the cell is the penetration of the plasma membrane lipid bilayer. First of all, the plasma membrane lipid bilayer is fairly impervious to most molecules of biologic and medical interest. Moreover, the fact that the lipid bilayer can vary significantly in terms of its polar lipid, protein, glycoprotein and carbohydrate composition from cell type to cell type poses a significant and arduous challenge in introducing various macromolecules into the cells in a systematic manner.
A number of chemical, physical and biological techniques have been devised for introducing macromolecules such as DNA and RNA into mammalian cells. The most widely used method encompasses lipid-mediated transfection which works well for some cell types but not others in particular primary cells and immune cells (Liu, D., Ren, T., and Gao, X., 2003, Curr. Med. Chem. Vol. 10, pp 1307-1315; Nicolazzi, C., Garinot, M., Mignet, N., Scherman, D. and Bessodes, M. 2003, Curr. Med. Chem., Vol. 10, pp 1263-1277). In addition, a number of cells are extremely sensitive to lipid-mediated transfection and there can be significant degree of cytotoxicity associated with this method. Amine-based transfection is another technique that has been utilized for transfection (Blagbrough, I. S., Geall, A. J. and Neal, A. P., 2003, Biochem. Soc. Trans, Vol. 31, pp 397-406). However, it is also prone to the same challenges as lipid-mediated transfection. Another method for introduction of macromolecules into mammalian cells is based on microinjection where a specially designed microcapillary needles are used in conjunction with a micromanipulator apparatus and a microscope (Lamb, N. J., Gauthier-Rouviere, C. m and Fernandez, A. 1996, Front Biosci. Vol. 1, pp 19-29). While, this method is fairly efficient specially for hard to transfect cells such as primary cells and neurons (Washbourne, P. and McAllister, A. K., 2002, Curr Opin Neurobiol. Vol. 12, pp 566-573), its wide scale use has been restricted due to its technical hurdle as well as its throughput and at this moment in time is certainly not feasible for genome-scale procedures. Viral-mediated gene transfer is another method of introducing DNA and RNA into mammalian cells (Hapala, I., 1997, Crit. Rev. Biotechnol. Vol. 17, pp 105-122). Several different systems such as adenovirus and vaccinia virus systems have been successfully used for efficient transfection of mammalian cells, especially neuronal cells. While viral system may work efficiently at the level of single genes, it utility at genome-wide scale is significantly compromised due to the time it takes for construction of viral vectors and for obtaining optimal viral titers for infection. In summary, while a number of procedures have been optimized for transfection and some have been used for genome-wide introduction of genes and RNAi into mammalian cells, none of the procedures discussed are optimal for high throughput, efficient and reproducible introduction of macromolecules into mammalian cells. There is a need to develop a novel, efficient, and reproducible method that can introduce and deliver molecules to cells.
Subsequent to cellular transfection of macromolecules by various means, the effect of the macromolecule(s) are analyzed in the appropriate cellular context by different end-point assays. These end-point assays can only provide information about the cellular effects of the macromolecule transfected only at a specific time point. In addition, these assays typically are applicable to only a single cellular event that is to be analyzed.
U.S. Pat. No. 6,686,193 disclosed instrumentation and methods for screening drug candidate compounds with activity against ion channel targets. The method included modulating the transmembrane potential of host cells in a plurality of sample wells with a repetitive application of electrical fields so as to set the transmembrane potential to some target levels. A number of devices were disclosed. However, the devices had limitations in delivering effective electrical fields to population of cells in the wells.
In a publication by Burnett et al (“Fluoresence Imaging of Electrically Stimulated Cells”, by P Burnett, J K Robertson, J M Palmer, R R Ryan, A E Dubin and R A Zivin, in Journal of Biomolecular Screening, volume 8 (6), 2003, pp 660-667), the authors described some preliminary results obtained from devices that were designed to supply electrical stimuli to population of cells. Using a digital fluorescent microscope, changes in voltage-gated ion channel activity were monitored. As an example, a device with an interdigitated electrode fingers were used to electrically stimulate cells. However, these techniques have not been successfully coupled to a real time electronic cell sensing system.